The use of biologically active substances, such as proteins, is enhanced when such substances are covalently immobilized, i.e., coupled as ligands, onto supports. Separation techniques such as affinity chromatography are based on the ability of the coupled ligand to bind specific, targeted biologically active substances from a mixture of other materials. Common examples of affinity chromatography techniques include the binding of immunoglobulins using coupled proteins and the binding of antigens using coupled antibodies.
Successful ligand coupling is based on two factors: quantity immobilized and quality of immobilization. Quantity immobilized, expressed as density per unit volume of support, is an indicator of the amount of ligand coupled regardless of the quality of that immobilization. In fact, most protein coupled in highly dense regions of a support is biologically inactive. That is a waste.
Quality of immobilization, expressed as bound specific biological activity, is an indicator of the amount of ligand coupled onto a support in a manner that causes the ligand to retain its biologically activity. Maximizing bound specific biological activity is desirable. However, there must be enough ligand density to achieve practical utility.
The optimal condition in ligand coupling would be the maximum amount of ligand that is coupled with maximum bound specific biological activity. That results in optimal ligate binding or functional efficiency of the coupled ligand on the support.
Thus, for purposes of this application, "functional efficiency" means the combination of acceptable quantity of ligand coupling with acceptable bound specific biological activity.
Most ligand candidates are large molecules that have specific conformations necessary to retain biological activity. In the case of antibody binding of antigen, low antigen binding efficiencies have been attributed to the concerted actions of surface density of antibody, multi-point attachment of antibody to porous supports, undesirably restrictive conformations imposed by covalent attachment, steric effects, and orientation effects. See Velander et al., "The Use of Fab-Masking Antigens to Enhance the Activity of Immobilized Antibodies", Biotechnology and Bioengineering, Vol. 39, 1013-1023 (1992) which discloses how enhanced functional efficiency was achieved when the Fab portion of a monoclonal antibody was masked with synthetic antigens prior to covalent immobilization of the antibody on the support, followed by unmasking.
Enhanced bound specific biological activity was disclosed in PCT Publication WO 92/07879 (May 14, 1992) by the use of polyanionic salts in concentrations of greater than about 0.5M during covalent immobilization of biologically active materials to azlactone functional supports. Preferably, in addition to polyanionic salts, amounts of azlactone quencher were added during covalent immobilization to compete with the biologically active material during covalent immobilization.
Notwithstanding these advances in the field of ligand coupling technique, there remains the problem of optimizing functional efficiency of coupling expensive and precious biologically active substances as ligands to supports.
The problem of optimal functional efficiency has not been solved by others. For example, U.S. Pat. No. 4,968,742 (Lewis et al.) uses an elaborate, stepwise method to couple ligands involving derivatizing a polymer with an activating agent to introduce a couplable functional group, with the derivatization performed in the presence of a blocking agent which is reactive with the same functionality on the polymer as the activating agent, in order to control the number of couplable functional groups for covalent immobilization of ligand.
U.S. Pat. No. 4,839,419 (Kramer et al.) discloses a method for adsorbing a protein onto a support and then crosslinking the protein to the support where the reaction conditions for coupling do not differ from those of the adsorption.
U.S. Pat. No. 4,775,714 (Hermann et al.) discloses a two-step process of immobilization of biologically efficient compounds on a carrier involving the steps of hydrophobic interaction and covalent immobilization. Examples 5-7 therein disclose the stepwise addition of a solution of inorganic salt, in a concentration of between 0.5 M to 3.0 M, to a reaction vessel containing the biologically efficient compound and the carrier, followed by a slow reaction (40 hours at 40.degree. C. under moderate shaking) in order to produce an immobilized, biologically active compound.
There have been efforts to experimentally determine how coupled protein was distributed to activated porous supports. See Stage et al. Biochimica et Biophysica 343, 382-391, (1974), where uniform distribution was reported for immunoglobulins coupled to cyanogen bromide-activated Sepharose branded beads. See also, Lasch et al. Eur. J. Biochem. 60, 163-167 (1975) which reported that uniform distributions of ferritin were found, except when the CNBr activation of Sepharose branded beads was very high (&gt;50 mg/ml) and/or when coupling efficiency was higher than 90%. Thus, non-uniform distribution resulted from high coupling efficiencies.
While non-uniform distribution of immobilized enzymes on porous supports has been studied using alterations in reaction conditions, and, in some cases, found preferable, others have warned of the possibility that non-uniform antibody immobilization on Sepharose branded beads was responsible for the loss of binding activity with increase of average antibody density. See Tharakan et al. J. Chrom. 522, 153-162 (1990).
Another sought to improve the performance of an immobilized enzyme by "kinetic control" during the immobilization by using salt concentration and time to control the enzyme distribution to an ionic support and chemically couple with a reagent added to fix this distribution. See Borchert et al. Biotechnology and Bioengineering (26)7, 727-736 (1984). Another has studied reaction conditions and proposed a restriction effect at openings of pores in the porous support that could prevent further protein from being coupled to the support. See Clark et al. Biotechnology and Bioengineering 26(8) 892-900 (1984).
Generally, the art has found that one can achieve coupling efficiency by reacting at conditions to achieve a high coupling capacity at the expense of loss of bound specific biological activity. Alternatively, the art has found that one can achieve high bound specific biological activity with lower coupling efficiency (because the total amount of ligand coupled is lowered.) In both of these circumstances, the reaction conditions were not altered during the reaction process except as described in U.S. Pat. No. 4,775,714 (Hermann et al.) in Examples 5-7. In that circumstance, salt was added stepwise after the ligand solution and the support were combined but before the reaction commenced for 40 hours at 40.degree. C.